1. Pharmacologic Inhibition of IκB Kinase Activates Immediate Hypersensitivity Reactions in Mice 
Dai Miyazaki, Sachiko Mihara, Koudai Inata, Shin-ichi Sasaki, Takeshi Tominaga, Keiko Yakura, Waka Ishida, Atsuki Fukushima, Yoshitsugu Inoue 
The American Journal of Pathology 
Volume 183, Issue 1, Pages (July 2013)
DOI: /j.ajpath
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
Aggravation of allergen-induced mast cell degranulation and systemic surge of inflammatory cytokines by IKK inhibition. A: Allergen-sensitized mice were injected i.v. with IKK inhibitor peptide 1 day before the challenge by allergen. Allergen-induced mast cell degranulation was significantly exacerbated by the injection of 20 μg per mouse of IKK inhibitor peptide. ∗P < 0.01, ∗∗P < n = 12 per group. B: Surge of systemic inflammatory cytokines after IKK inhibition. Serum samples from allergen-challenged mice were analyzed for inflammatory cytokine induction by cytokine array analysis. IKK inhibitor treatment induced an array of inflammatory cytokines, including IL-1β, IL-9, CCL2, and CCL3. ∗P < 0.05, ∗∗P < n = 9 per group. Data are expressed as means ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

3. Figure 2
Network analysis of allergen-induced ocular immediate hypersensitivity reactions. A set of 509 allergen-induced genes (fold induction >1.4) were complemented with IL-9 and CCL2 and were analyzed for network generation of biological processes. The highest significant six networks (P < 1 × 10−29) are summarized as merged networks. NFkB (complex), IKK family, and IL-1 are positioned in the center of the network and are highlighted in yellow. Nodes of IL-9 and CCL2 are positioned downstream of IL-1.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

4. Figure 3
Role of IL-1 in the aggravation of allergen-induced mast cell degranulation by IKK inhibition. A: Naive mice were injected i.v. with IKK inhibitor peptide of the indicated amount. Serum samples collected after 24 hours were tested for systemic levels of caspase 1 using ELISA. ∗P < 0.05. n = 6 per group. B: Role of IL-1 in aggravated mast cell activation by pharmacologic inhibition of IKK. Allergen-induced mast cell degranulation was significantly enhanced after 20 μg per mouse of IKK inhibitor peptide treatment. This enhancement was blocked by the administration of IL-1RA. ∗∗P < n = 12 per group. C: IL-1RA–treated mice after IKK inhibitor peptide administration were challenged with allergen, and eyelid skin was Giemsa stained after 90 minutes. Allergen-induced mast cell granulation (asterisks) was enhanced in releasing mast cell granules (arrows) by IKK inhibitor peptide treatment and was negated by IL-1RA treatment. Original magnification, ×1000. Data are expressed as means ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

5. Figure 4
IL-1β induction and exacerbation of allergen-induced mast cell degranulation by B220+ B cells after IKK inhibition. A: Digested tissue-derived cells were separated based on surface markers of CD4, CD8, CD11b, PDCA-1, or B220. The cells were screened for IL-1β secretion after IKK inhibitor peptide treatment. Dose-responsive secretion of IL-1β was observed for the B220+ B-cell fraction. n = 4 per group. B: IKK inhibitor peptide–treated B220+ B cells were co-cultured with IgE-sensitized mast cells, and allergen-induced mast cell degranulation was evaluated by determining β-hexosaminidase release. IKK inhibitor peptide–treated B220+ B cells significantly enhanced allergen-induced mast cell degranulation, whereas B220− cell populations (including mixed subsets of CD4+ or CD8+ or CD11b+ or PDCA-1+) did not. ∗P < n = 6 per group. C: Isolated tissue-resident mast cells were co-cultured in Transwells (Corning Inc., Corning, NY) with B cells treated with IKK inhibitor peptide. Allergen-induced β-hexosaminidase release from mast cells was significantly enhanced by co-culturing with IKK inhibitor–treated B cells and suppressed by IL-1RA. ∗P < 0.01, ∗∗P < n = 6 per group. Data are expressed as means ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

6. Figure 5
Role of B cells in aggravated mast cell activation by pharmacologic inhibition of IKK. Mice were passively sensitized with ragweed-specific IgE and induced for allergic conjunctivitis by allergen instillation. Allergen-induced mast cell degranulation was significantly enhanced after treatment with IKK inhibitor peptide or isohelenin. This enhancement was not observed in B-cell–deficient mice [Ighmtm1Cgn targeted mutation (μMT)]. Wt, wild type. ∗P < 0.05, ∗∗P < n = 14 per group. Data are expressed as means ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

7. Figure 6
Secretion of IL-1β in B cells after IKK-β inhibition. IKK-β inhibition by IKK-β inhibitor VIII resulted in the secretion of IL-1β (A) and CCL2 (C) from B cells isolated from the spleen. In contrast, IKK-α inhibition by siRNA transfection did not appreciably induce IL-1β (B) or CCL2 (D). ∗P < 0.05. n = 6 per group. Data are expressed as means ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

8. Figure 7
Enhanced NF-κB binding activity of NF-κB family subunits in B cells after IKK inhibitor treatment. A: Reduced degradation of IκBα by IKK inhibition. IκBα degradation of B cells isolated from the spleen was inhibited by 0.02 μg/mL IKK inhibitor peptide or 10 nmol/L IKK-β inhibitor VIII. Whole cell extracts of B cells were assayed for IκBα by Western blot analysis. Treatment by IKK inhibitor peptide or IKK-β inhibitor VIII significantly elevated the IκBα levels. B: Nuclear extract from B cells were profiled for binding activity of subunits of the NF-κB family, p52, RelB, p50, and p65 to NF-κB–responsive consensus sequence. IKK inhibitor peptide, 0.02 μg/mL, significantly elevated the binding activity of p52, RelB, p50, and p65. IKK-β inhibitor VIII (10 or 100 nmol/L) elevated the binding activities of p52, RelB, and p50. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < n = 4 per group. Data are expressed as means ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

9. Figure 8
Role of IKK-α in NF-κB promoter activation and enhanced recruitment of IKK-α to NF-κB–responsive IκBα promoter in B cells after IKK/IKK-β inhibition. B cells isolated from the spleen were inhibited for IKK/IKK-β activity by 0.02 μg/mL IKK inhibitor peptide or 10 nmol/L IKK-β inhibitor VIII. A: IKK/IKK-β inhibition induced IκBα mRNA in B cells by real-time PCR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B: ChIP assay showed an enhanced recruitment of IKK-α but not IKK-β to NF-κB–responsive IκBα promoter in splenic B cells after IKK/IKK-β inhibition. C: B cells were transiently transfected with NF-κB reporter plasmid and treated with 10 nmol/L IKK-β inhibitor VIII. IKK-β inhibitor VIII significantly activated NF-κB promoter as detected by luciferase assay. The enhanced promoter activity was suppressed by IKK-α inhibition by cotransfection of IKK-α siRNA. ∗P < n = 4 per group. Data are expressed as means ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

10. Figure 9
Role of IKK-α and B cells in the aggravated immediate hypersensitivity reaction after IKK inhibition. Mice were passively immunized with ragweed-specific IgE and challenged with ragweed allergen. Allergen-induced mast cell degranulation was significantly enhanced after IKK inhibitor treatment. To specifically target IKK-α in B cells, mice were systemically injected with IKK-α siRNA-conjugated anti-B220 antibody. B-cell–targeted inhibition of IKK-α negated the aggravation of mast cell activation by IKK inhibition. ∗P < 0.05, ∗∗P < n = 12 per group. Data are expressed as means ± SEM.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2013 American Society for Investigative Pathology Terms and Conditions